PROJECT SUMMARY_PROJECT 2 The design of valid clinical trials involving botanical dietary supplements requires the optimization of the trial product such that it contains sufficient levels of the active compounds. The identification of these active compounds therefore has to be a part of any serious study of botanicals. It is also recognized that the overall activity profile of a botanical may be mediated by multiple active compounds, which can act independently, additively, synergistically or be antagonistic to each other. While functional effects of a botanical can be demonstrated in vivo, initial studies on the identification of active compounds and their interaction is most efficiently evaluated using in vitro, ex vivo, or moderate throughput in vivo approaches. These also have the advantage of being mechanism based. Centella asiatica (CA) and Withania somnifera (WS) have been shown to affect cognition, sleep and mood and in this project, we will use primary neuron assays, brain slices, and Drosophila models to explore mechanisms and active compounds of CA and WS. Primary neurons will be used to address effects of CA and WS and their compounds on neuronal health by measuring neuronal arborization, antioxidant responses, reactive oxygen species, and mitochondrial function. In addition, we will determine effects on vascular tone and resilience to vasoconstriction in mouse brain slices to test whether these extracts promote vascular health. Drosophila models will be used to identify compounds that improve the age-related decline in locomotion and reactivity. We will also test effects on a described depression-like state in Drosophila, using courtship as an assay, and we will measure Serotonin levels in neuronal subpopulations identified as mediating this depression-like state. Furthermore, Drosophila will be used to determine effects on sleep patterns, which are also altered by age in flies and mammals. The cellular and molecular pathways regulating sleep are well known in Drosophila and we can therefore also investigate whether CA and/or WS promotes healthy sleep patterns by altering neuronal activity and neurotransmitter signaling in the neuronal populations that promote or suppress sleep. Besides Serotonin and Aceteylcholine, this includes GABA. To expand these studies to the mouse model, we will measure neuronal activity in mouse brain slices and address whether treatment with CA and/or WS affects GABA signaling (using GABA inhibitors). Together, these studies will identify active compounds in CA and WS and they will show whether they support health and resilience by promoting cognition, sleep and/or mood. In addition, they will provide insights into mechanism that may mediate these effects, like improving neuronal activity, decreasing oxidative stress and/or promoting mitochondrial function. Future studies can then confirm these effects in an in vivo mouse model and eventually provide the basis for testing active compounds in humans.